am-356 and Brain-Neoplasms

Cannabinoids have been associated with tumor regression and apoptosis of cancer cells. Recently, we have shown that R(+)-methanandamide (R(+)-MA) induces apoptosis of H4 human neuroglioma cells via a mechanism involving de novo expression of the cyclooxygenase-2 (COX-2) enzyme. The present study investigated a possible involvement of a mitochondrial-driven pathway in this process.. Cell death was determined by the WST-1 cell viability test, and changes in apoptotic parameters [i.e., release of mitochondrial cytochrome c, activation of caspases, cleavage of poly(ADP-ribose) polymerase (PARP)] were detected by Western blotting.. H4 cells treated with R(+)-MA showed typical signs of mitochondrial apoptosis, i.e., release of mitochondrial cytochrome c into the cytosol and activation of initiator caspase-9. Moreover, activation of the executor caspase-3 was observed following cannabinoid treatment. Cells were fully protected from apoptotic cell death by the caspase-3 inhibitor Ac-DEVD-CHO, indicating a crucial role for caspase-3 activation in R(+)-MA-elicited apoptosis. Furthermore, cleavage of the caspase-3 target protein PARP was registered. All of the aforementioned effects were substantially reduced by the selective COX-2 inhibitor celecoxib (1 muM) at a pharmacologically relevant, nonapoptotic concentration.. R(+)-MA-induced apoptosis is mediated via a mitochondrial-dependent pathway that becomes activated, at least in part, through up-regulation of the COX-2 enzyme.

Topics: Animals; Apoptosis; Arachidonic Acids; Blotting, Western; Brain Neoplasms; Caspase 3; Caspase 9; Caspases; Celecoxib; Cell Survival; CHO Cells; Cricetinae; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cytochromes c; Glioma; Humans; Mitochondria; Poly(ADP-ribose) Polymerases; Pyrazoles; Signal Transduction; Sulfonamides

Cannabinoids induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells via a pathway independent of cannabinoid- or vanilloid receptor activation. The underlying mechanism was recently shown to involve increased synthesis of ceramide, which in turn leads to activation of p38 and p42/44 mitogen-activated protein kinases (MAPKs). The present study investigates a possible contribution of membrane lipid rafts to cannabinoid-induced COX-2 expression. To address this issue, we tested the influence of methyl-beta-cyclodextrin (MCD), a membrane cholesterol depletor, on COX-2 expression by the endocannabinoid analogue R(+)-methanandamide (R(+)-MA). Incubation of H4 cells with MCD was associated with a loss of lipid raft integrity and a substantial inhibition of R(+)-MA-induced COX-2 expression and subsequent formation of prostaglandin E2. Moreover, MCD was shown to suppress signal transduction steps upstream to COX-2 induction by R(+)-MA. Accordingly, the cholesterol depletor suppressed R(+)-MA-induced formation of ceramide as well as phosphorylation of p38 and p42/44 MAPKs. Together, our results suggest that R(+)-MA induces COX-2 expression in human neuroglioma cells via a pathway linked to lipid raft microdomains.

Topics: Arachidonic Acids; beta-Cyclodextrins; Blotting, Western; Brain Neoplasms; Cell Line; Cell Line, Tumor; Ceramides; Cholesterol; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Glioma; Humans; Isoenzymes; MAP Kinase Signaling System; Membrane Microdomains; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Topics: Arachidonic Acids; Brain Neoplasms; Cannabinoid Receptor Modulators; Cannabinoids; Cyclooxygenase 2; Endocannabinoids; Gene Expression Regulation, Enzymologic; Glioma; Humans; Isoenzymes; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Prostaglandin-Endoperoxide Synthases; Tumor Cells, Cultured

Cannabinoids affect prostaglandin (PG) formation in the central nervous system through as yet unidentified mechanisms. Using H4 human neuroglioma cells, the present study investigates the effect of R(+)-methanandamide (metabolically stable analogue of the endocannabinoid anandamide) on the expression of the cyclooxygenase-2 (COX-2) enzyme. Incubation of cells with R(+)-methanandamide was accompanied by concentration-dependent increases in COX-2 mRNA, COX-2 protein, and COX-2-dependent PGE(2) synthesis. Moreover, treatment of cells with R(+)-methanandamide in the presence of interleukin-1beta led to an overadditive induction of COX-2 expression. The stimulatory effect of R(+)-methanandamide on COX-2 expression was mimicked by the structurally unrelated cannabinoid Delta(9)-tetrahydrocannabinol. Stimulation of both COX-2 mRNA expression and subsequent PGE(2) synthesis by R(+)-methanandamide was not affected by the selective CB(1) receptor antagonist AM-251 or the G(i/o) protein inactivator pertussis toxin. Enhancement of COX-2 expression by R(+)-methanandamide was paralleled by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK. Consistent with the activation of both kinases, R(+)-methanandamide-induced COX-2 mRNA expression and PGE(2) formation were abrogated in the presence of specific inhibitors of p38 MAPK (SB203580) and p42/44 MAPK activation (PD98059). Together, our results demonstrate that R(+)-methanandamide induces COX-2 expression in human neuroglioma cells via a cannabinoid receptor-independent mechanism involving activation of the MAPK pathway. In conclusion, induction of COX-2 expression may represent a novel mechanism by which cannabinoids mediate PG-dependent effects within the central nervous system.

Topics: Analgesics, Non-Narcotic; Arachidonic Acids; Blotting, Western; Brain Neoplasms; Cannabinoid Receptor Modulators; Cell Line; Central Nervous System; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Dronabinol; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Glioma; Humans; Imidazoles; Interleukin-1; Isoenzymes; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Pertussis Toxin; Phosphorylation; Piperidines; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Pyridines; Receptors, Cannabinoid; Receptors, Drug; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tumor Cells, Cultured; Virulence Factors, Bordetella